Progress Report for Neural Differentiation Unit For the past year, the Neural Differentiation Unit has made continuous progress.We also initiated collaborations with other groups within NINDS and NIH. Specific aim 1: In vitro neurogenesis and development modeling using neural cultures derived from human adult peripheral CD34+ cells. We optimized our protocol and we can now derive both neural stem cells (iNS) and iPSC cells simultaneously from 10 ml of blood. We have published a paper on this technique and another video protocol paper is in press. Specific aim 2:Study the effect of inflammation on oligodendrocyte progenitor cells.We found that T cells released VEGF to increase OPC proliferation through VEGF receptor type 2 (VEGF-R2).These findings may have potential impact on treating T cell inflammatory-related neurodegenerative disorders. A manuscript based on these findings has been submitted for publication. Specific aim 3:Study the effect of HERV K on pluripotent stem cell development. We have found that HERV K components, gag, env and pol expression were increased in iPSCs but diminished rapidly after differentiation. We found Herv-K Env interacted with cell membrane protein CD98HC, which plays an important role in maintaining stemness and cell morphology. Inhibition using siRNA or antibody against Env resulted in stem cell morphological changes. These finding suggested an important role HERV-K may have played in stem cell development. A paper is in prepared for publication based on these findings. Specific aim 4: Study the mechanisms of neurodegenerative disorders. We are generating more iPS/iNS from cells of ALS (3 cell line) and PLS (6 cell lines) in collaboration with Dr. Mary Key Floeter. We are differentiating the cells to motoneurons and will compare their gene expressions with normal controls using microarray. We are also providing human neural cells for Dr. Roche to study the possible role of neuroligin-4 in Autism. Our unit also deriving iNS cells from patient samples from Undiagnosed disorders program and differentiate neural cells to study disease pathogenesis. Progress Report Summary for NTDU Within the NTDU group, we have utilized a number of automated, medium throughput assays in the past year, to screen small compound collections against rat mixed cortical cultures, human neural stem cells and human NSC-derived neurons along with some astrocyte cultures.Weve utilized live cell imaging based assays with rat hippocampal/cortical neurons, as well as developed fluorescence and high resolution phase contrast assays to probe high content imaging endpoints. These assays have been employed in multiple screens, working with numerous investigators (Rao, Malik-CRM, Pant, Major and others). We have continued to generate probe or tool compounds in our chemistry labs to facilitate screening and mechanistic studies in our research projects. We produce and purify a number of recombinant proteins, such as the HIV Tat protein for our own studies and provide that to extramural researchers around the world for their studies. With the Section of Insections of the Nervous System (SINS), we have continued to work on three research projects. We have found that the HERVKenv protein is toxic to mixed rat hippocampal cultures, human neural stem cells and human NSC-derived neurons. The HIV Tat antagonist program has progressed with the generation of LTR-luciferase constructs that are more sensitive to Tat in eliciting Tat-dependent selective LTR activation, and upon incorporation into a stable cell line, will facilitate screening of the 14,000 compound Maybridge HitCreator collection for new lead Tat inhibitors. The studies incorporating HIV Tat with A&#61538;1-40 continue, as we evaluate whether these neurotoxic effects result from cell membrane interactions or from intracellular means. The NTDU has completed a number of studies in collaboration with the Center for Regenerative Medicine, developing a high throughput assay to discover compounds that were toxic to neural stem cells, but not neurons or astrocytes. The screen of 2000 compounds identified more than 50 compounds toxic to human NSCs but not mixed rat cortical neurons. One class of compounds that we identified as being particularly toxic to human but not rat neural cells, was cardiac glycosides, like digoxin, oleandrin, lanatoside C and ouabain. We continue to work with Dr. Harish Pant and his research team to characterize the neuroprotective actions of the Cdk5 modulator TFP-5. In another series of experiments, treatment of neural stem cells with TFP-5 had no significant effect on NSC proliferation and self renewal. However, we observed that treatment of NSCs with TFP-5 resulted in an accelerated neurite growth and branching. We continue to work with Gene Majors lab to develop a hybridization ELISA assay to quantitate the JCV antisense oligonucleotide levels in serum and CSF. Likewise, we are beginning to work with David Sibleys group to characterize the dopamine D3 receptor agonists that they have identified, using our live cell imaging platforms for quantitative analysis. The Clinical Proteomics Unit goal is to initiate discussions with collaborators in order to provide guidance on aspects of experimental design that are critical to producing high quality analyses and statistically meaningful results. We utilize multi-dimensional liquid chromatography as a primary means of reducing sample complexity but also utilize affinity and electrophoretic techniques when indicated. A significant portion of the Clinical Proteomics Unit efforts focus on methods development. We are developing an automated workflow for cerebrospinal fluid (CSF). Our strategy is to use affinity chromatography to deplete albumin and IgGs from CSF prior to hydrolyzing the remaining proteins in an immobilized enzyme reactor and capture the resultant peptide mixture. Individual depleted CSF samples are labeled with an isobaric mass tags, pooled with several other samples and analyzed by tandem mass spectrometry. Bioinformatic analysis then affords both protein identification and relative quantitation. This list of candidate protein biomarkers will be rapidly quantified in large numbers of clinical samples by Parallel Reaction Monitory (PRM), a high resolution form of Multiple Reaction Monitoring (MRM). Proteomic analysis of non-depleted CSF resulted in 887 protein IDs, while our method resulted in 630 proteins IDs of which 341 were unique to the depleted CSF dataset. Importantly, Gene Ontology analysis indicates a majority of the unique IDs were related to neuronal structure and function.